Comparison of Hybribio-H13 and Hybrid Capture® 2 human papillomavirus tests for detection of CIN2+ and CIN3+

Abstract Introduction. Low-cost, accurate high-risk HPV tests are needed for cervical cancer screening in limited-resource settings. Objective. To compare the performance of the low-cost Hybribio-H13 test with the Hybrid Capture® 2 to detect cervical intraepithelial neoplasia grade 2 or 3 (CIN2 and CIN3). Materials and methods. Archived baseline samples tested by the Hybrid Capture® 2 from women of the ASCUS-COL trial, aged 20 to 69 years, with biopsy-colposcopy directed diagnosis of CIN2+ (n = 143), CIN3+ (n = 51), and < CIN2 (n = 632) were blindly tested by the Hybribio-H13 test. Results. The relative sensitivity of the Hybribio-H13 test versus the Hybrid Capture® 2 for detecting CIN2+ was 0.89 (90% CI = 0,80-0,98; NIT = 0,66), and for CIN3+ was 0,92 (90% CI = 0,85-0,98; NIT = 0,35). Relative specificity was 1.19 (90% CI = 1.05-1.33; NIT < 0.00001). In the analysis restricted to women older than 30 years, the relative sensitivity of the Hybribio-H13 for CIN3+ was marginally below unity (ratio = 0.97; 90% CI = 0.95-0.99), and the specificity remained higher than the Hybrid Capture® 2 test. Conclusion. The Hybribio-H13 test was as specific as the Hybrid Capture® 2 for detecting CIN2+ or CIN3+ but less sensitive. Considering these results and the young age of the population recruited for screening because of ASCUS cytology, we suggest our results warrant the evaluation of the Hybribio-H13 for screening cervical cancer, especially in the evaluated population.

In 2020, there were 604,127 new cases and 341,831 deaths due to cervical cancer.Around 90% of these cases and deaths occur in Asia, Africa, Latin America, and the Caribbean regions (1).Human papillomavirus (HPV) is the cause of virtually all cervical cancers, with HPV types 16 and 18 accounting for approximately 70% of the cases (2).Prophylactic vaccination against HPV-16 and HPV-18 provides more than 90% protection against infection and associated high-grade lesions -like cervical intraepithelial neoplasia (CIN) grade 2 or 3 (CIN2 and CIN3)-or cancer (3).However, because currently implemented HPV vaccines do not eliminate the risk of cervical cancer, early detection remains a public health need.
Cytology-based screening is associated with an important reduction in the incidence and mortality of cervical cancer, especially in high-income countries but it has not achieved that impact in low-middle income countries.The main reason is the low sensitivity of cytology, which requires repeated testing that hinders required access to regular screening and follow-up to gynaecological management of positive results (4).Human papillomavirus testing has a sensitivity of around 100% to detect cervical high-grade lesions and has a high negative predictive value allowing the extension of screening to every five years.Other important attributes include automation, high reproducibility, and faster turnover of results than cytology (5).Therefore, HPV testing is an alternative currently available for cervical screening, especially in low-middle income countries where performer-dependable method implementation has been challenging.However, HPV testing has not been widely implemented in routine healthcare services of these countries.Most of the current HPV tests are expensive and require advanced equipment (6).
The Hybribio-13 HPV test from Hybribio (Hybribio Biotechnology Limited Corp., Hong Kong, China), hereafter referred to as H13, is a low-cost test based on a quantitative polymerase chain reaction (qPCR) that detects as a pool the 18,31,33,35,39,45,51,52,56,58,59, and 68 genotypes in cervical exfoliates (7).The H13 test does not require complex infrastructure and is robust, with an easy interpretation of results obtained in about three hours.
Although these results suggest that H13 or H14 might be attractive for cervical cancer screening in low-resource settings given its low cost, no study has compared the performance of H13 or H14 to reference standards in samples of women from low-middle income countries.In this secondary analysis of the phase III randomized controlled ASC-US trial (9) we present a head-tohead comparison of the H13 assay with the reference QIAGEN© HC2 HPV DNA to the detect CIN2+ and CIN3+ in 842 women participating in this trial.

Study design and population
Samples for this study were selected from the ASCUS-COL trial.The ASC-US-COL is a three-arm, non-blinded, parallel-group pragmatic trial.Women aged 20 to 69 years (n = 2,661) with first-time presence of atypical squamous cells of undetermined significance (ASC-US) detected by cytology in the last two years were flagged in routine screening services and randomly allocated to receive immediate colposcopy (IC arm; n = 882), repeat cytology at 6 and 12 months (RC arm; n = 890), or an HPV test within two months of recruitment (HPV arm; n = 889).Colposcopy and biopsies, according to clinician judgment, were recommended for all women in the IC arm, for women with a repeat ASC-US or worse (ASC-US-positive) cytology in the RC arm, and a high-risk HPV test for women in the HPV arm.Hybrid Capture 2 HPV DNA test (HC2, Qiagen™, Germantown, USA) was conducted at the laboratory of infection and cancer at the Universidad de Antioquia.All women received invitations, and 80% (n = 2,132) attended the exit visit after 24 months of follow-up, which included high-risk HPV and cytology tests (9).All women positive for either test were referred to a certified, well-trained colposcopy specialist using a standardized and controlled protocol of biopsy sampling.After the end of the study, two blinded accredited experts confirmed the histopathological diagnoses of 1,407 women with at least one histological (n = 1.327) or endocervical (n = 80) diagnosis record, and the baseline samples of women in the IC and RC arms were tested for high-risk HPV by HC2 (Qiagen™) (supplementary figure 1).ASCUS-COL is registered with ClinicalTrials.gov(NCT02067468).

Selection of participants for sub-study HC2 vs H13 comparison
Women identified after the end of the ASCUS-COL trial with biopsycolposcopy-directed, adequate diagnosis, and enough remaining archived baseline samples in Specimen Transport Medium™ (Qiagen™) for further testing (n = 1,348) were considered eligible for this study.We included all women diagnosed with CIN2+ (n = 197) and a representative sample of agematched women (n = 645) with a final negative or CIN1 histological diagnosis, as shown in supplementary figure 1.The residual content of the specimen transport medium tube, used for the HC2 HPV DNA testing of the collected samples at the recruitment visit, was used for the H13 testing.Data collection and testing of the reference standard (histopathological diagnosis) and comparator test (HC2) were conducted before the index test (H13).The HC2 and H13 assays, as well as the verification of the histological diagnoses, were conducted independently and blindly.

Qiagen™ HC2 HPV DNA test
This test is based on a DNA-RNA hybridization that identifies a pool of 13 high-risk HPV genotypes (16,18,31,33,35,39,45,51,52,56,58,59, and 68).Cervical cells were collected from women with a cytobrush (HC cervical sampler) and transferred to a tube containing 1 ml of Specimen Transport Medium™.The HC2 HPV DNA testing (Qiagen™, Gaithersburg, MD, USA) was performed according to the manufacturer's instructions at the HPV laboratory of the Grupo de Infección y Cáncer at the Universidad de Antioquia (Medellín, Colombia).Relative light unit values -higher than one-were considered positive.

HybriBio (H13) DNA extraction and testing
The DNA for the HybriBio-13 test was extracted from the denatured residual content of the specimen transport medium tube used for the HC2 HPV DNA test by a standard protocol (10).Briefly, each specimen was digested for two hours at 55 °C with 200 µg of proteinase K per ml and 1% Laureth-12.The samples were heated to 95 °C for 10 minutes to denature the residual protease.After precipitation with 5M ammonium acetate and 70% ethanol, DNA was washed, dried, and resuspended in 100 µl of TE buffer (10 mM TRIS + 0.1 mM EDTA) and frozen at -30 °C until shipped at room temperature to the Laboratory of Translational Genomics of the National Cancer Institute (NCI/ NIH, Bethesda, MD, USA), where testing was performed.
The H13 test is a real-time qPCR assay that uses specific primers and probes targeting the HPV E6 and the human β-globin genes.One of the probes is labeled with 6-carboxyfluorescein (FAM) fluorescent dye for the detection of a pool of 13 high-risk HPV genotypes and the other with (6-carboxy-4´,5´-dichloro-2´,7´-dimethoxyfluorescein, HEX) fluorescent dye and shows human β-globin gene working as an internal control.The reagents' volume and input DNA were modified from those described in the manufacturer's instructions.The final volume was 11 µl, including 8.75 µl of PCR Master Mix kit, 0.25 µl of DNA Taq Polymerase, and 2 µl of the sample DNA.We have previously shown that this modification results in the minimum assay volume required with equivalent results (11).We used the positive and negative controls included in the H13 kit.A Ct value lower than or equal to 40 was the threshold considered as a positive result.Negative samples with no positive signal in the internal control were excluded from the analysis.The real-time instrument used was a Roche LightCycler 480 II.

Sequencing of BSGP5+/6+ amplimers
To determine the HPV type of discordant samples (positive HC2/negative H13, n = 97, and negative HC2/positive H13, n = 29), DNA purified from exfoliates as described above was amplified with BSGP5+/6+ primers and the amplicon was sequenced with Sanger.The conserved BSGP5+/6+ primer pair amplifies a region of 150 bp of the L1 gene containing unique sequences that distinguish HPV genotypes (12,13).The sequences were used as a query for screening the GenBank database (www.ncbi.nlm.nih.gov) with BLAST Software 1. HPV types were assigned when we found a match between the 150 bp interprimer region and an HPV sequence in the GenBank.

Sample size
We excluded 16 invalid samples -six negatives, seven CIN1, and three CIN2-because of unusual signals in the cellular internal control of the H13 test.The final analysis included 826 women.Based on the recommendations of Meijer et al. that at least 60 samples should be analyzed to assess whether a candidate test has a sensitivity for CIN2+ not less than 90% of that of HC2 (14), our study included 194 samples with CIN2+ for a power of 99.6% and 51 samples with CIN3+ for a power of 92.7%.Non-inferiority test (NTI) of H13 to HC2 concerning clinical specificity for < CIN2 was assessed in 632 cervical samples of women who did not have histologically confirmed CIN2+ with a power of 90%.

Statistical methods
Sensitivity and specificity with corresponding 95% confidence intervals were estimated to detect CIN2+ or CIN3+ using < CIN2 (without neoplasia or CIN1) as disease-free categories.The McNemar test (McN) was applied to compare the differences between matched proportions.A matched noninferiority test (NIT) with a 90% relative sensitivity threshold and a 98% relative specificity threshold was applied to compare the clinical performance of the H13 to the HC2.Statistical significance for both statistics (McN and NIT) was set at 0.05.All analyses were conducted using the Stata 13 software (StataCorp LLC, Texas, USA).

Ethical approval
ASCUS-COL complied with Colombian Resolution 8430 of 1993 to conduct studies in humans and followed the CIOMS guidelines (15).The ethics committees for human experimentation of the Sede de Investigación Universitaria (SIU) (Resolution 08-036-171) and the Escuela de Medicina (Resolution 004/2008) from the Universidad de Antioquia approved this study.Participants signed written informed consent, including authorization to use their samples and data for future research.

Results
Specimens from 842 women, collected and previously tested with the HC2 at the enrolment visit, were selected from the 2,661 participants of the ASCUS-COL study with adequate histological diagnosis and retested by the H13 (figure 1).Sixteen (1.9%) samples of these residual specimens tested invalid with H13 and were excluded from further analysis, resulting in 826 samples analyzed by both the HC2 and H13 tests.The clinical features of the studied population are summarized in table 1.All women had an ASCUS pap smear for the first time at the screening visit.Most women were under 40 years old (75%), around half started regular sex between 16 and 19 years old, and 54% had between three and four lifetime sexual partners.Slightly more than half of the women included in this analysis had a definitive histological diagnosis at a six-month follow-up (57%).The number of histological diagnoses in the 842 included participants were: 506 women without cervical lesions, 139 with CIN1, 146 with CIN2, 47 with CIN3, and four with cancer.HC2 and H13 tests were positive in 389 (60%) and 335 (52%) of the 645 women with < CIN2; and in 182 (92%) and 160 (81%) of the 197 CIN2+ cases, respectively.For the CIN3+ threshold, HC2 and H13 tests were positive in 48 (94%) and 44 (86%) of 51 women with CIN3+, respectively (supplementary table 1).

Discussion
In this study, we compared the clinical accuracy of the Hybribio-H13 test to the Hybrid Capture™ 2 (Qiagen™).Due to the matched design with samples tested with both H13 and HC2, we could calculate non-inferiority statistics.Samples were tested immediately after collection by the HC2 in Colombia and shipped to the USA, where testing by H13 was conducted using the minimum assay volume.Under these conditions, the H13 test did not conform to the acceptable standards of clinical performance for sensitivity to detect CIN2+ or CIN3+ but complied with the acceptable standards for specificity to detect < CIN2, overall, and in women of 30 years or older.
Currently, few studies properly comparing the clinical performance of H13 with standard reference tests have been published in peer-reviewed literature.A recent study described the clinical performance between the H13 and the HC2 tests in 373 samples from North America.The H13 correctly identified 94% of the HC2 HPV-positive CIN2+ cases and 88% of the HC2 HPV-negative cases (7).Likewise, in our study, The H13 identified 156 of the 180 (87%) HC2 CIN2+-positive cases and 224 of the 249 (90%) HC2 HPV-negative < CIN2 cases.In contrast to that description reporting 143/516 (28%) equivocal results, in our hands, the H13 test was highly robust, as the proportion of samples with equivocal results was very low (16/842; 1.9%).3. Relative sensitivity for CIN2+ and CIN3+, and relative specificity for < CIN2 and CIN3+ for the H13 and the HC2 test comparison a p for the McNemar test to set differences between matched proportions.b p for the non-inferiority test.A matched non-inferior statistic (ni) with a 90% relative sensitivity threshold and 98% relative specificity threshold was used to compare clinical performance of Hybribio-H13 (H-13) to Hybrid Capture™ 2 (HC2) tests.Sixteen (six negative biopsies, seven CIN1, and three CIN2) samples, tested invalid by H13 test, were excluded from the analysis.CIN2: cervical intraepithelial neoplasia grade 2; CIN3: cervical intraepithelial neoplasia grade 3; HC2: Hybrid Capture™ 2 (Qiagen™); H13: Hybribio-H13 (Hybribio Biotechnology Limited Corp) CI: Confidence interval; TN: true negative; FP: false positive; TP: true positive; FN: false negative We excluded 16 samples (six negative biopsies, seven for CIN1, and three for CIN2) from analysis due to invalid results for H13 test.In our study, HC2 samples were processed immediately after collection, and shortly after, manually extracted DNA was shipped at room temperature to the USA for the H13 testing.We cannot exclude the possibility that the differences between tests could be because of the modifications to the manufacturer's instructions.Therefore, our results must be interpreted within the scope of this limitation.This study is the first in the international literature that presented a head-to-head comparison of the H13 assay with the reference Hybrid Capture™ 2 (Qiagen™) for the detection of CIN2+ and CIN3+ in a group of samples that allowed the performance of robust statistical tests with adequate power.
In this study, we included women with first-time ASCUS cytology at routine screening visits to healthcare services, 75% between 20 and 39 years of age.Under these conditions, the H13 test did not conform to the acceptable standards of clinical performance for sensitivity to detect CIN2 or CIN3+ but conformed for the specificity to detect < CIN2.In the analysis restricted to women aged 30 years or more, the relative sensitivity of the H13 for CIN3+ was marginally below unity (ratio = 0.97; 90% CI = 0.95-0.99),and the specificity remained higher than the HC2.
In conclusion, this study is the first to compare head-to-head the performance of the H13 test with a reference test such as the Hybrid Capture™ 2 (Qiagen™).The H13 test was as specific but less sensitive than HC2 to detect CIN2+ or CIN3+.Considering these results and the young age of the population recruited for screening due to ASCUS in the cytology, we suggest the H13 test is useful for screening cervical cancer, especially in women over 30 years who are subjected to screening with HPV tests according to Colombian clinical practice guidelines, and that these data contribute to the use of the H13 test as a screening method., or 68) and some low-risk HPV types.Sixteen samples (six negative biopsies, seven CIN1, and three CIN2) tested invalid by H13 and were excluded from the analysis.Genotype 66 was not considered since it was not found in either of the two tests.

Figure 1 .
Figure 1.Flowchart showing the process from the sample panel collation and HPV testing to the final endpoint ascertainment of diseased and non-disease groups.STM: Specimen transport medium.
) Laboratory of Translational Genomics, USA 842 hydrid capture 2 (HC2) results (0 invalids) University of Antioquia, Colombia Randomly selected samples from women with ASC-US at screening with an adequate histological diagnosis

Table 2 .
Specificity and sensitivity of HC2 and H13 HPV tests for the detection of CIN2+ and CIN3+

table 1 .
Neoplasia per age range, and HC2 and H13 test results according to histological diagnosis Supplementary